The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. α, β1, and γ2 subunits). Membranes were incubated with [35S]guanosine 5′-(3-O-thio)triphosphate (GTPγS) ± an agonist, and the amount of radiolabel bound to the α subunit was quantitated following immunoprecipitation. When expressed without receptor (but with β1γ2), the G protein subunits αz, α12, and α13 did not bind appreciable levels of [35S]GTPγS, consistent with a minimal level of GDP/[35S]GTPγS exchange. In contrast, the subunits αs and αq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPγS to αz but not to α12, α13, or αs. Binding to αz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPγS binding to αz and more robust increases in binding to αq, α12, and α13. Binding of [35S]GTPγS to αs was strongly enhanced only by the activated β1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.